The present invention relates to a screening method for SREBP (sterol regulatory element binding protein) pathway-specific inhibitors. The present invention also relates to therapeutic agents for hyperlipemia, arterial sclerosis, obesity or cancer containing an SREBP pathway-specific inhibitor selected by said screening method.
Cholesterol is an essential substance for living bodies as a component of cell membranes or a precursor for syntheses of steroid hormones or bile acids. However, excessive accumulation thereof is fatal for cells, and therefore, homeostatic maintenance of cellular cholesterol level is a very important physiological mechanism. At the level of living bodies, excessive accumulation of cholesterol is also known to cause various diseases such as hyperlipemia or atherosclerosis. The results of a recent large-scale clinical test on cholesterol-lowering drugs showed that the mortality of patients of cardiac diseases is improved by lowering serum cholesterol level, revealing the importance of the maintenance of a proper cholesterol level in living bodies (Scandinavian Simvastatin Survival Study Group, Lancet (1994) 344:1383-1389; Gotto, A. M., Am. J. Med. (1998) 104:6S-8S).
Homeostatic maintenance of cellular cholesterol level is known to occur at various stages such as the level of transcription, translation, enzyme, or the like. Recent discovery of factors involved in transcriptional control of a group of cholesterol synthases or low-density lipoprotein (LDL) receptors dramatically accelerated an understanding of the mechanism of homeostatic regulation of cholesterol at transcriptional level.
LDL receptors or cholesterol synthases such as HMG-CoA reductase are directly responsible for the maintenance of cholesterol level as a gate for cholesterol uptake into cells or a source of newly synthesized cholesterol. It is well known that expression of these factors is feedback-regulated depending on the cholesterol level. Recently, sterol regulatory element binding proteins (SREBPs) that are transcriptional activation factors binding to a sterol regulatory element (SRE) in the promoter domain of these genes was shown to be involved in said regulation (Brown, M. S. and Goldstein, J. L. Cell (1997) 89:331-340).
SREBP has two isoforms of closely related structures expressed from different genes, SREBP-1 and SREBP-2. Both are expressed as membrane proteins spanning the membrane twice with both ends oriented to the cytosol in the endoplasmic reticulum membrane or nuclear membrane. Upon decrease of cellular cholesterol level, the envelope protein SREBP is thought to be cleaved with proteases in two steps to release the activated N-terminal DNA-binding domain from the membrane, which migrate into the nucleus to activate transcription of target genes (FIG. 1). The protease-catalyzed two-step cleavage mechanism has been mostly unknown. It has been shown that the first step of cleavage is sensitive to cholesterol while the second step (site 2) of cleavage automatically occurs as a result of the first step (site 1) of cleavage.
In M19, which is a mutant cell line of Chinese hamster ovary (CHO)-K1 cells (ATCC: CRL-9618) with lowered cholesterol synthesis and LDL receptor activities (Hasan, M. T. et al., Somat. Cell Mol. Genet. (1994) 20:183-194), a study has shown that no cleavage occurs at site 2 of SREBP (Sakai, J. et al. Cell (1996) 85:1037-1046), and a gene for a protease responsible for cleavage at site 2, S2P has been cloned by complementation using said cell line (Rawson et al., Mol. Cell (1997) 1:47-57). The SREBP sequence cleaved with S2P has not been determined but it is known to lie near the N-terminal first membrane-spanning domain.
On the other hand, the cleavage at site 1 is known to occur between Leu and Ser, but the enzyme per se has not been known. Another mutant cell line of CHO-K1 cells, 25RA (Chang, T. Y. and Limanek, J. S., J. B. C. (1980) 255:7787-7795) is resistant to 25-hydroxycholesterol and does not lead to cell death by cholesterol overload, differing from CHO-K1 cells. On the basis of dominant sterol resistancy of 25RA cells, an SREBP cleavage-activating protein (SCAP) was cloned by using an expression library prepared with 25RA cells. An analysis of the SCAP gene of 25RA cells and CHO-K1 cells revealed that one of two point mutations on the SCAP gene of 25RA cells involved a change from aspartic acid to asparagine at position 443 to enhance SREBP cleavage activity at site 1 without being regulated by sterols. The other mutation was a silent mutation. In other words, cholesterol synthesis or LDL receptor activity is not lowered by sterols in 25RA cells carrying mutations on SCAP because SREBP is cleaved at site 1 even in the presence of excessive sterols, revealing that SCAP has an important function in the SREBP cleavage regulation by cholesterol.
In this way, establishment of mutant cells derived from CHO-K1 promoted an understanding of the mechanism of transcriptional control of cholesterol metabolism. This mechanism is thought to be common to mammalian cells such as human or murine cells.
Target genes for SREBPs as reported include enzymes for the cholesterol synthesis system such as HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, squalene synthase, as well as enzymes for the fatty acid synthesis system such as acetyl CoA carboxylase or fatty acid synthase, enzymes for the triglyceride synthesis system such as glycerol-3-phosphate acyltransferase, and the SREBP-2 gene itself (FIG. 1). This suggests that inhibition of the SREBP pathway may result in inhibition of synthesis of lipids such as cholesterol. This possibility was experimentally verified using sterols such as 25-hydroxycholesterol in cultured cells. However, direct demonstration on individual level has not been made. Moreover, recent reports have shown diversity of target genes for SREBPs, suggesting that the SREBP pathway may be involved in various physiological phenomena, as will be described later.
Despite attempts to develop various cholesterol synthesis inhibitors, many of them have not been successful because of toxicity or other reasons. HMG-CoA reductase inhibitors are among rare cases of success, but their effect to lower serum cholesterol was not evaluated as sufficient (Illingworth, D. R., Am. J. Cardiol. (1993) 72:54D-58D). At present, HMG-CoA reductase inhibitors and bile acid reabsorption inhibitors are mainly used for therapy of hyperlipemia. Both drugs decrease serum cholesterol by lowering cholesterol in the liver and activating low-density lipoprotein (LDL) receptors. However, the effect is attenuated by cholesterol uptake into the liver following activation of LDL receptors and no potent cholesterol-lowering effect can be obtained in any case, because these drugs rely on an indirect mechanism of lowering cholesterol in the liver to activate LDL receptors. On the other hand, the results of a large-scale clinic test on serum cholesterol-lowering drugs based on said inhibitors indicated benefits of lowering serum cholesterol in patients with cardiac diseases and raised expectations for more potent cholesterol-lowering drugs.
From this background, we assumed that lipid-lowering agents inhibiting lipid synthesis such as cholesterol synthesis by inhibiting the SREBP pathway may be very useful therapeutic agents for hyperlipemia and also useful for therapy of arterial sclerosis. In view of the fact that intermediate products or metabolic products of the cholesterol synthesis system expressed at a level controlled by SREBPs have been reported to be ligands for PPARs (peroxisome proliferator activated receptors), orphan receptors (Forman, B. M. et al., Cell (1995) 81:687-693; Janowski, B. A. et al., Nature (1996) 383:728-731; Lala, D. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1997) 94:4895-4900), it is useful to use said mechanism for therapy of diseases caused by a decrease in these molecules. Inhibition of the SREBP pathway also seems to be useful for therapy of obesity, since PPARxcex3 was shown to be an important determinant for differentiation to adipocytes and activation of PPAR (Peroxisome Proliferator Activated Receptor) potentially induced by activation of SREBP was also reported (Kim, J. B. et al., Proc. Natl. Acad. Sci. U.S.A. (1998) 95:4333-4337). Inhibition of the SREBP pathway also seem to be applicable as anticancer agents since some intermediate products of the cholesterol synthesis system are used to modify Ras or other genes playing an important role in cell growth. Thus, inhibition of the SREBP pathway may be promising for therapy of various diseases.
However, any convenient screening method for inhibitors of this pathway has not been known up to the present. Noting the above-mentioned 25RA and M19 cells as well as DM7 cells carrying mutations of said two cell lines (Hasan, M. T. et al., Somat. Cell Mol. Genet. (1994) 20:481-491), we examined the potential of a screening system for SREBP pathway-specific cholesterol synthesis inhibitors using these cells having mutations in the SREBP pathway and designed a convenient screening system.
The present invention provides a screening method for SREBP pathway-specific inhibitors using a mutant cultured cell.
A preferred first embodiment of the screening method of the present invention is a screening method for SREBP pathway-specific inhibitors, comprising assaying both cholesterol synthesis and LDL receptor activities using a cell in which SREBP cleavage activating protein (SCAP) has lost response to sterols.
A preferred second embodiment of the screening method of the present invention is a screening method for sterol-like SREBP pathway-specific inhibitors using a combination of a cell in which SCAP has lost response to sterols and a cell in which SCAP has not lost response to sterols.
A preferred third embodiment of the screening method of the present invention is a screening method for SREBP pathway-specific inhibitors using a combination of a cell lacking SREBP Site 2 protease (S2P) and a S2P-carrying cell.
A preferred fourth embodiment of the screening method of the present invention is a screening method for SREBP pathway-specific inhibitors using a cell in which a chimeric gene of a reporter gene and the gene encoding the C-terminus of SREBP has been expressed.
A preferred fifth embodiment of the screening method of the present invention is a screening method for sterol-like SREBP pathway-specific inhibitors, comprising screening for SREBP pathway-specific inhibitors using a cell in which a chimeric gene of a reporter gene and the gene encoding the C-terminus of SREBP has been expressed, and then screening for sterol-like SREBP pathway-specific inhibitors using a cell in which SCAP has lost response to sterols and the same chimeric gene has been expressed.
A preferred sixth embodiment of the screening method of the present invention is a screening method for S2P-specific inhibitors using a cell in which a chimeric gene of a reporter gene and the gene encoding the stretch from the first transmembrane domain of SREBP to the cleavage site with SREBP Site 1 protease (S1P) has been expressed.
The present invention also provides a vector obtained by inserting a chimeric gene of a reporter gene and the gene encoding the C-terminus of SREBP into a reporter gene expression vector.
The present invention also provides a vector obtained by inserting a chimeric gene of a reporter gene and the gene encoding the stretch from the first transmembrane domain of SREBP to the cleavage site with SREBP Site 1 protease (S1P) into a reporter gene expression vector.
The present invention also provides SREBP pathway-specific inhibitors obtained by the screening method of the present invention.
The present invention also provides therapeutic agents for hyperlipemia containing thus obtained SREBP pathway-specific inhibitors.
The present invention also provides therapeutic agents for arterial sclerosis containing thus obtained SREBP pathway-specific inhibitors.
The present invention also provides therapeutic agents for obesity containing thus obtained SREBP pathway-specific inhibitors.
The present invention also provides therapeutic agents for cancer containing thus obtained SREBP pathway-specific inhibitors.